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Establishment Of A Rat Acute Traumatic Deep Vein Thrombosis Model And Femoral Vein Wall Gene Expression Study

Posted on:2006-11-29Degree:DoctorType:Dissertation
Country:ChinaCandidate:B WangFull Text:PDF
GTID:1104360155476957Subject:Surgery
Abstract/Summary:PDF Full Text Request
Objectives: Through establishing a rat model of acute traumatic limbs deep vein thrombosis (DVT) and detecting femoral vein wall genes expressional variations at crucial phases by gene chip technology, to explore the mechanisms of acute traumatic limbs DVT occurrence or not, severity, thrombi resolution or insolution.Methods: 1.250 SD rats were divided randomly into two parts: control group (group A, n=20) and experimental groups (n=230). According to different phases after model being produced, the experimental groups were divided into 6 groups: trauma instant group (B), thrombosis prophase group (C), thrombosis crest-time group (D), thrombi resolution group (E), thrombi insolution group (F), non-thrombosis group (G).2. Rats were not anesthetized in model producing process. After regional sterilizing, 1cm long inguinal groove internal incision was adopted to expose proximal femoral vein. 1.5cm isolated femoral vein was clamped at three points separately with 12.5mm mosquito-hemostatic forceps, once each point; the clamping strength was fastening one barb of hemostatic forceps, lasting 3 seconds each time, then the incision was sutured, no drainage was set. Rat hibateral posterior limbs were fixed with hip spica casts except for group A and B. At different phases after model being produced, color and swelling extent of both feet were observed by gross observation.3. Vessel specimens resecting: rats were anesthetized with 3% pentobarbital sodium (1 ml/kg, intraperitoneal injection), supine position fixation, sterilization, exposing hibateral femoral veins. In group A, 4.5cm femoral vein and related main tributaries were resected. In group B, at 0.5h; group C, at 2.5h; group D, at 25h; group E, F, G, at 72h after model being produced, the same region vascular tissue was also resected separately. Part of the proximal vessel separated for histological analysis, the other vessels were rinsed by 0.9% physiological saline; the vessel involving thrombi should be cleaned up. The vessel specimens were put into nitrogen canister in less than 30 seconds after ex vivo, which would be used for total mRNA extraction.4. Through TRIzol method,total mRNAs of femoral vein specimens from the 7 phases were extracted separately. All total mRNAs samples were checked by agarose gel electrophoresis, cRNA probes preparation, hybridization, washing, staining and scanning were performed orderly to finish array detecting. All data were analyzed and summarized.Results: 1. In group A, B, C, no rat presented thrombosis. In the 190 rats of group D, E, F, G, total 5 rats died. From 2.5h to 72h after model being built, 149 rats presented thrombosis, 36 were no thrombosis, 64 presented thrombi resolution and 37 presented thrombosis insolution.2. The 7 total RNA samples were approved to be high qualities without degradation.3. The hybridization signal intensity of arrays was satisfactory, the results were reliable.4. In the 15923 rat genes which can be detected by Affymetrix RAT 230A microarray, 2504 displayed differential expression: 1187 were up-regulated, 1338 were down-regulated. At thethrombosis crest-time, 489 genes were up-regulated, 696 were down-regulated. In the non-thrombosis state, 135 genes were up-regulated, 278 were down-regulated. At the thrombi resolution phase, 573 genes were up-regulated, 642 were down-regulated. At the thrombi insolution phase, 584 genes were up-regulated, 681 were down-regulated. In the 2504 differential expressional genes, 208 genes were significantly differential expression (Signal log2ratio>3.2orS3.2).5. At different phases, in the genes related to blood coagulation-anticoagulation, 3 were up-regulated, 1 was down-regulated. In the genes related to fibrolysis-antifibrolysis, 8 were up-regulated, 2 were down-regulated. In the genes related to immune response, 35 were up-regulated and 2 were down-regulated. In the genes related to inflammatory response, 59 were significantly up-regulated.6. In the down-regulated genes, the number of genes presented in 4 or more than 4 phases was 358, in which, 20 significantly down-regulated genes at trauma instant were associated with muscular movement and metabolic regulations. And 4 unknown function genes (Rn.41395^ Rn.22504^ Rn.l5517> Rn.19158) presented coexpression.7. hi the up-regulated genes, the number of genes presented in 4 or more than 4 phases was 564, in which, the genes at trauma instant were mainly associated with immune and inflammatory responses.Conclusions: 1. A novel rat model of acute traumatic DVT can be established through clamping femoral vein with hip spica cast fixation. This method is simple and easy to perform, which also has a good repeatability and a stable result. The pathological process of DVT occurrence or not, thrombi resolution or insolution is simulated much better in this model.2. In the process of thrombosis occurrence or not, thrombi resolution or insolution, femoral vein wall differential expression genes are mainly associated with blood coagulation, anticoagulation, fibrolysis, antifibrolysis, immune response, inflammatory response.3. The genes related to fibrolysis-antifibrolysis are up-regulation generally at several phases, in which, the genes related to antifibrolysis displayed a much more obviously differential expression.4.4 significant down-regulation unknown function genes (Rn.41395N Rn.22504> Rn.15517^ Rn. 19158) may be associated with muscular movement and metabolic regulation.5. Trauma instant may be not the beginning of inflammatory response but may have already be the expressional climax of genes associated with inflammatory response.
Keywords/Search Tags:Acute, Traumatic, Deep vein thrombosis, Animal model, Gene chip
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